The carboxyl-terminal lobe of Hsc70 ATPase domain is sufficient for binding to BAG1.

The molecular co-chaperone BAG1 and other members of the BAG family bind to Hsp70/Hsc70 heat shock proteins through a conserved BAG domain that interacts with the ATPase domain of the chaperone. BAG1 and other accessory proteins stimulate ATP hydrolysis and regulate the ATP-driven activity of the chaperone complexes. Contacts are made through residues in helices alpha2 and alpha3 of the BAG domain and predominantly residues in the C-terminal lobe of the bi-lobed Hsc70 ATPase domain. Within the C-terminal lobe, a subdomain exists that contains all the contacts shown by mutagenesis to be required for BAG1 recognition. In this study, the subdomain, representing Hsc70 residues 229-309, was cloned and expressed as a separately folded unit. The results of in vitro binding assays demonstrate that this subdomain is sufficient for binding to BAG1. Binding analyses with surface plasmon resonance indicated that the subdomain binds to BAG1 with a 10-fold decrease in equilibrium dissociation constant (K(D) = 22 nM) relative to the intact ATPase domain. This result suggests that the stabilizing contacts for docking of BAG1 to Hsc70 are located in the C-terminal lobe of the ATPase domain. These findings provide new insights into the role of co-chaperones as nucleotide exchange factors.

Structural analysis of BAG1 cochaperone and its interactions with Hsc70 heat shock protein.

BAG-family proteins share a conserved protein interaction region, called the 'BAG domain', which binds and regulates Hsp70/Hsc70 molecular chaperones. This family of cochaperones functionally regulates signal transducing proteins and transcription factors important for cell stress responses, apoptosis, proliferation, cell migration and hormone action. Aberrant overexpression of the founding member of this family, BAG1, occurs in human cancers. In this study, a structure-based approach was used to identify interacting residues in a BAG1--Hsc70 complex. An Hsc70-binding fragment of BAG1 was shown by multidimensional NMR methods to consist of an antiparallel three-helix bundle. NMR chemical shift experiments marked surface residues on the second (alpha 2) and third (alpha 3) helices in the BAG domain that are involved in chaperone binding. Structural predictions were confirmed by site-directed mutagenesis of these residues, resulting in loss of binding of BAG1 to Hsc70 in vitro and in cells. Molecular docking of BAG1 to Hsc70 and mutagenesis of Hsc70 marked the molecular surface of the ATPase domain necessary for interaction with BAG1. The results provide a structural basis for understanding the mechanism by which BAG proteins link molecular chaperones and cell signaling pathways.

Ligand responsiveness in human prostate cancer: structural analysis of mutant androgen receptors from LNCaP and CWR22 tumors.

Androgen receptors (ARs) belong to the family of hormone receptors that are ligand-dependent transcription factors. Endocrine therapy provides effective treatment for prostate cancer until mutations arise that alter the ligand responsiveness of AR. In this study, structural models were developed for the functional domains of human AR by homology modeling from crystal structures of closely related nuclear receptors. These models were used to locate the sites of two frequently occurring mutations in prostate cancer. The substitutions that develop in LNCaP (threonine-->alanine at residue 877) and CWR22 (histidine-->tyrosine at residue 874) tumor cell lines are both located on helix 11 that forms part of the ligand-binding pocket. However, the results suggest that these mutations influence ligand responsiveness by completely different mechanisms. Residue 877 contacts the ligand directly, and substitution at this site alters the stereochemistry of the binding pocket. Thus, the LNCaP mutation apparently broadens the specificity of ligand recognition. In contrast, residue 874 is located down the helical axis, projects away from the ligand pocket, and does not contact ligand. The side chain of residue 874 lies in a cavity between helices 11 and 12. Substitution of tyrosine for histidine 874 in CWR22 tumors may affect a conformational change of helix 12 and, thus, influence binding of coactivator proteins and their regulatory effect on transcriptional activation.

Site-selective control of the reactivity of surface-exposed histidine residues in designed four-helix-bundle catalysts.

BACKGROUND: The structure and function of native proteins often depend on the interplay between ionisable residues with physical properties that have been fine tuned by interactions with neighbouring groups. Here, we systematically vary the environment of histidines in designed helix-loop-helix motifs to modulate histidine pKa values and reactivities. RESULTS: 25 helix-loop-helix motifs were designed in which surface-exposed histidine residues were flanked by neutral, negatively charged and positively charged groups and the histidine's proximity to the hydrophobic core was varied. The 57 histidine pKa values were determined by 1H NMR spectroscopy and found to be in the interval 5.2-7.2 with changes ranging from a decrease of 1.3 pKa units to an increase of 0.7 pKa units compared with the pKa for an unperturbed histidine residue. CONCLUSIONS: A decrease in the pKa of His34 by 1.3 units was accomplished by placing it in close proximity to the hydrophobic core and flanking it by positively charged residues in positions (i, i + 3) and (i, i - 4). Flanking a histidine residue with a lysine or a histidine in positions (i, i + 3), (i, i + 4) or (i, i - 4) resulted in pKa depressions of approximately 0.5 pKa units per residue and additivity was observed. The increase of the histidine pKa by glutamate residues was the most efficient in position (i, i + 3), but less efficient in position (i, i + 4). These principles should be useful in the engineering of novel catalysts.